Validation, capillary columns

Flynn LR, Bass SW, Meierer RE. 1991. Headspace screening/capillary column GC/MS analysis for volatile organics validation studies and applications. Waste Testing and Quality Assurance 106-114. 

Liquid chromatography is now a mature technique. Instruments are reliable and increasingly computer assisted. Column-to-column reproducibility is ensured by most manufacturers. The quest for the universal detector is about to end with the advent of a sophisticated and miniaturized MS detector. Development of a method can be achieved in a rather short period with available software. The emphasis is on validation more than on how to handle it. Capillary columns are sure to improve, and the trend will be toward many parallel analyses. 

A stereoselective GC method for determination of etodolac enantiomers in human plasma and urine was first reported as a preliminary method [35], and then as a validated method [36]. Sample preparation involved addition of (S)-(+)-naproxen (internal standard) and sodium hydroxide to diluted plasma or urine. The samples were washed with diethyl ether, acidified with hydrochloric acid, and extracted with toluene. ( )-(+)-naproxen was used as a derivatizing agent to form diastereomeric derivatives of etodolac. The gas chromatograph system used in this work was equipped with fused-silica capillary column (12 m x 0.2 mm i.d.) coated with high-performance cross-linked methylsilicone film (thickness 0.33 pm) and a nitrogen-phosphorous detector. The operating conditions were injector 250°C detector 300°C column 100-260°C (32 °C/min). 

Equation (2) is valid for surface potential of up to 50 mV. For packed capillary columns, the dependence of electroosmotic flow velocity on operating parameters based on Eq. (2) was examined and it was found that electroosmotic flow velocity increases with column porosity, particle diameter, and concentration of bulk electrolyte [21],... 

Like gas chromatography, supercritical fluid chromatography can be practised both in packed and capillary column techniques. Observations made earlier under gas chromatography in regard to column performance of capillary columns compared with that of packed columns, are also valid for supercritical fluid chromatography. 

The analysis of the collected average shift samples was made with a method validated by us, based on NIOSH method 1600/1994 (National Institute for Occupational Safety and Health). After eluation of the activated charcoal with toluene the test was made with a gas chromatograph with mass selective detector Perkin Elmer and capillary column DB-5, 30 m long and thickness of the coating 0.25pm. Helium was used as carrier gas. Apparatus conditions injector temperature 250°C, detector temperature 250°C, carrier gas pressure - 7 psig. The quantitative assessment of the samples was performed after absolute calibration with standard solutions of carbon disulfide in toluene. The limit of detection of the method is 0.01 mg/m3 at 25dm3 air sample. 

Solid-phase micro-extraction (SPME) first became available to analytical researchers in 1989. The technique consists of two steps first, a fused-silica fiber coated with a polymeric stationary phase is exposed to the sample matrix where the analyte partitions between the matrix, and the polymeric phase. In the second step, there is thermal desorption of analytes from the fiber into the carrier gas stream of a heated GC injector, then separation and detection. Headspace (HS) and direct insertion (DI) SPME are the two fiber extraction modes, whereas the GC capillary column mode is referred to as in-tube SPME. The thermal desorption in the GC injector facilitates the use of the SPME technology for thermally stable compounds. Otherwise, the thermally labile analytes can be determined by SPME/LC or SPME/GC (e.g., if an in situ derivatization step in the aqueous medium is performed prior to extraction). Different types of commercially-avarlable fibers are now being used for the more selective determination of different classes of compounds 100 /rm polydimethylsiloxane (PDMS), 30 /rm PDMS, 7 /rm PDMS, 65 /rm carbowax-divinylbenzene (CW-DVB), 85 /rm polyacylate (PA), 65 /rm PDMS-DVB, and 75 /rm carboxen-polydimethyl-siloxane (CX-PDMS). PDMS, which is relatively nonpolar, is used most frequently. Since SPME is an equilibrium extraction rather than an exhaustive extraction technique, it is not possible to obtain 100% recoveries of analytes in samples, nor can it be assessed against total extraction. Method validation may thus include a comparison of the results with those obtained using a reference extraction technique on the same analytes in a similar matrix. 

The UV detector is also very frequently used in SFC, mainly in packed column SFC, because of problems in sensitivity with capillary columns. Since pure carbon dioxide shows little absorbance between 200 and 800 nm, UV detection in packed-column SFC is very sensitive. In general, the characteristics of UV detectors discussed in Section 12.2.6.3 are also valid for UV-SFC detectors, except that the latter must be equipped with a pressure-resistant flow cell. A normal UV detector flow cell in LC can resist a maximum pressure of ca. 7- 10 MPa. In SFC, the UV detector cell must be under the same separation conditions (temperature and pressure) as the column (if the pressure in the detector cell were lower, the fluid would loose its solvation power), so it should resist pres.sure < 40 MPa. 

The analysis of PCBs in various mediums, such as water, soils or oil, is a delicate operation that has been mastered. Gas chromatography with an electron capture detector, which can be coupled in problem cases with a mass spectrometer, is the most suitable method. This article briefly outlined the calculation methods available. The excellent performance of capillary columns call into question current testing standards, which warrant review and revision. Furthermore, the traditional sample preparation methods (i.e., PCB extraction, cleanup, concentration) are evolving and being simplified considerably with the introduction of a wide variety of very specific ready-to-use cartridges. Nevertheless, many tests remain to be performed in this area by laboratories already equipped for PCB analysis so that the effectiveness of their work can be validated. 

Packed columns will continue to be used for gas chromatographic methods that were validated on packed columns where time and cost of revalidation on capillary columns would be prohibitive. 

Few well characterized, validated methods are available for the determination of w-hexane in blood. A purge-and-trap method for volatiles has been developed and validated by researchers at the Centers for Disease Control and Prevention (CDC) (Ashley et al. 1992, 1994). Extension of the method to include /7-hexane should be possible. Current analytical methods utilize capillary GC columns and MS detection to provide the sensitivity and selectivity required for the analysis. Detection limits are in the low ppb range (Brugnone et al. 1991 Schuberth 1994). Headspace extraction followed by GC analysis has also been utilized for the determination of /7-hexanc in blood (Brugnone et al. 1991 Michael et al. 1980 Schuberth 1994) however, very little performance data are available. 

HPLC methods can usually be transferred without many modifications, since most commercially available HPLC instruments behave similarly. This is certainly true when the columns applied have a similar selectivity. One adaptation, sometimes needed, concerns the gradient profiles, because of different instrumental or pump dead-volumes. However, larger differences exist between CE instruments, e.g., in hydrodynamic injection procedures, in minimum capillary lengths, in capillary distances to the detector, in cooling mechanisms, and in the injected sample volumes. This makes CE method transfers more difficult. Since robustness tests are performed to avoid transfer problems, these tests seem even more important for CE method validation, than for HPLC method validation. However, in the literature, a robustness test only rarely is included in the validation process of a CE method, and usually only linearity, precision, accuracy, specificity, range, and/or limits of detection and quantification are evaluated. Robustness tests are described in references 20 and 59-92. Given the instrumental transfer problems for CE methods, a robustness test guaranteeing to some extent a successful transfer should include besides the instrument on which the method was developed at least one alternative instrument. 

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